The steady increase in the number of decoded and mapped genomic sequences from flora and fauna is an impressive demonstration how important DNA-techniques are nowadays. But not the mere sequencing of DNA is of importance. With increasing knowledge in the field of genomics and proteomics, the impact of specific effects, e.g., mutations, on the future of cells or organisms comes into the focus of scientists. Since on the one hand, the nucleic acids are often present in very small concentrations and, on the other hand, they are often found in the presence of many other solid and dissolved substances, e.g., after lysis of cells, they are difficult to isolate or to measure.
Diverse methods for the detection, analysis and quantitation by hybridization of the target nucleic acid with a detectable probe have been established (e.g., Southern hybridization, dot blotting, gel-assays, PCR).
The main tool of nucleic acid related work, e.g., for amplification of polymeric nucleic acids, is the polymerase chain reaction (PCR). In recent years the knowledge about and the applications of PCR were noticeably expanded.
A PCR procedure consists in general of three steps: sample preparation, amplification, and product analysis. Although these steps are usually performed sequentially, amplification and analysis can occur simultaneously. DNA dyes or fluorescent probes can be added to the PCR mixture before amplification and used to analyze PCR products during amplification. The concurrent amplification and analysis of the sample within the same tube without changing the instrument reduces sample handling time, and minimizes the risk of product contamination for subsequent reactions. This approach of combining amplification with analysis has become known as “real time” PCR (U.S. Pat. No. 6,174,670).
Other possible amplification reactions are the Ligase Chain Reaction (LCR, Wu, D. Y., and Wallace, R. B., Genomics 4 (1989) 560-569; and Barany, F., Proc. Natl. Acad. Sci. USA 88 (1991) 189-193; U.S. Pat. No. 5,494,810); Polymerase Ligase Chain Reaction (Barany, F., PCR Methods Appl. 1 (1991) 5-16); Gap-LCR (PCT Patent Publication No. WO 90/01069; U.S. Pat. No. 6,004,286); Repair Chain Reaction (European Patent Publication No. 439 182 A2); 3SR (Kwoh, D. Y., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177; Guatelli, J. C., et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; PCT Patent Publication No. WO 92/08808); and NASBA (U.S. Pat. No. 5,130,238). Further, there are strand displacement amplification (SDA, U.S. Pat. No. 5,270,184; U.S. Pat. No. 5,455,166), transcription mediated amplification (TMA) and Q-beta-amplification (for a review see, e.g., Whelen, A. C., and Persing, D. H., Annu. Rev. Microbiol. 50 (1996) 349-373; Abramson, R. D., and Myers, T. W., Curr. Opin. Biotechnol. 4 (1993) 41-47), as well as isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN, Shimada, M., et al., Rinsho Byori. 51 (2003) 1061-1067) and cascade rolling circle amplification (CRCA, Thomas, D. C., et al., Arch. Pathol. Lab. Med. 123 (1999) 1170-1176).
For the above mentioned nucleic acid technique synthetic (deoxy)-oligonucleotides which have been provided with a detectable label are necessary, e.g., to carry out a broad spectrum of diverse molecular biological and molecular diagnostic methods.
Methods for the synthesis of single stranded oligonucleotide and oligonucleotide analogue sequences are known from the art (e.g., Oligonucleotide Synthesis: A Practical Approach, Gait, ed., IRL Press, Oxford (1984); Kuijpers, W. H. A., et al., Nucleic Acids Research 18 (1990) 5197-5205; Dueholm, K. L., J. Org. Chem. 59 (1994) 5767-5773; Agrawal, S. (ed.), Methods in Molecular Biology, volume 20).
The first effective and widely applicable method for the synthesis of oligo- and polynucleotides was the phosphotriester method (see, e.g., Letsinger, R. L., et al., JACS 91 (1969) 3360-3365). In this method the phosphate backbone of the synthesized polynucleic acid is already present. To prevent side reactions like branching during the synthesis reactive groups were protected with, e.g., the beta-cyanoethyl group or the ortho-chlorphenyl group. As activator for the coupling step mesityl sulfonyl chloride and mesityl sulfonyl nitrotriazole have been used.
Synthetic (deoxy)-oligonucleotides are usually prepared on a solid phase with the aid of phosphoramidite chemistry. Glass beads having pores of a defined size (abbreviated in the following as CPG, controlled pore glass) are usually used as the solid phase. The first monomer is bound to the support via a cleavable group such that the oligonucleotide can be set free by cleavage of this group after the solid phase synthesis is completed. The first monomer additionally contains a protected hydroxyl group, whereas dimethoxytrityl (DMT) is usually utilized as the protective group. The protective group can be removed by acid treatment. Then 3′-phosphoramidite derivatives of (deoxy)-ribonucleosides that are also provided with a DMT protective group are coupled in a cyclic process to each successive reactive group after is has been freed of the DMT protective group.
According to the prior art so-called trifunctional support materials are used to prepare oligonucleotides that are labeled at the 3′ end. For this a trifunctional spacer with two reactive hydroxyl groups and an additional reactive group, preferably an amino group, is firstly prepared. After introducing a DMT protective group on a hydroxyl group, the detectable label is coupled to the reactive amino group of the trifunctional spacer in a second step of the synthesis. However, alternatively the detectable label is not only coupled to the trifunctional spacer via a reactive amino group but also via a third hydroxyl group or an SH group (U.S. Pat. No. 5,451,463, WO 92/11388). In a third step the trifunctional spacer is bound via its hydroxyl group that is still free to the linking group of the solid phase material that is provided with a cleavable bond.
Alternatively the detectable label is not coupled until after the actual oligonucleotide synthesis (U.S. Pat. No. 5,141,837). However, since this requires multiple independent coupling reactions, such a production process is laborious, costly and cannot be automated.
Labeled phosphoramidites, in which the marker group is linked to the phosphoramidite via a C3-12 (C3-C12) linker, are usually used to synthesize oligonucleotides labeled at the 5′ end.
Hence detectable labels can also be introduced internally by the phosphoramidite strategy (Wojczewski, C., et al., Synlett 10 (1999) 1667-1678). The same trifunctional spacers can be used for this as for the synthesis of CPG materials. Instead of binding one of the hydroxyl groups to the solid phase, this hydroxyl group is converted into a phosphoramidite in this process. The resulting phosphoramidite can be used for oligonucleotide synthesis like a standard amidite. In principle such phosphoramidites can also be used for internal labeling by replacing a standard nucleoside phosphoramidite by a fluorophore-labeled phosphoramidite during the synthesis cycle. However, it is preferably used for 5′ labeling since internal labeling interrupts the base pairing in the strand.
Oligonucleotides provided with a fluorescent label such as fluorescein are often used in molecular biology, such as for the real-time measurement of PCR reactions (WO 97/46707). The fluorescent dyes can be coupled to, e.g., the amino group of the trifunctional spacer in different ways according to the prior art.
On the one hand the fluorescent dye, which can itself optionally be provided with cleavable protective groups for protection during the oligonucleotide synthesis, is reacted in the form of an isothiocyanate with the amino group to form a thiourea bond. In an alternative process the N-hydroxy-succinimide ester (NHS-ester) of a fluorophore-carboxylic acid is reacted with the free amino group of the spacer to form an amide bond. Alternatively the linker is terminated with a carboxyl group and is then reacted with an aminomodified label.
Beside these chemical methods for the preparation of labeled oligonucleotides enzymatic methods are available. For example a terminal label can be introduced using the enzyme terminal deoxynucleotidyl transferase which introduces an additional nucleotide at the end of an existing polydeoxynucleotide chain. This enzymatically introduced nucleotide bears the signal entity (see, e.g., EP 0122614).
The great success of real-time methods is closely linked to the detection or change of a reporter signal. This signal change evolves from the interaction of the probe molecule with the target molecule.
Monitoring fluorescence during each cycle of PCR initially involved the use of ethidium bromide (Higuchi, R., et al., Bio/Technology 10 (1992) 413417; Higuchi R., et al., Bio/Technology 11 (1993) 1026-1030; U.S. Pat. No. 5,994,056). In that system fluorescence is measured once per cycle as a relative measure of product concentration. Ethidium bromide detects double stranded DNA; if the template is present, fluorescence intensity increases with temperature cycling. Other fluorescent systems have been developed that are capable of providing additional data concerning the nucleic acid concentration and sequence.
In kinetic real time PCR, the formation of PCR products is monitored in each cycle of the PCR. The amplification is usually measured in thermocyclers which have additional devices for measuring fluorescence signals during the amplification reaction. In general, there exist different formats for real time detection of amplified DNA, of which the following are well known and commonly used in the art:
DNA binding dye format: Since the amount of double stranded amplification product usually exceeds the amount of nucleic acid originally present in the sample to be analyzed, double-stranded DNA specific dyes may be used, which upon excitation with an appropriate wavelength show enhanced fluorescence only if they are bound to double-stranded DNA. Preferably, only those dyes may be used which like Sybr Green I, for example, do not affect the efficiency of the PCR reaction (U.S. Pat. No. 6,174,670).
All other formats known in the art require the design of a fluorescent labeled hybridization probe which only emits fluorescence upon binding to its target nucleic acid.
TAQMAN probe: A single-stranded hybridization probe is labeled with two components. When the first component is excited with light of a suitable wavelength, the absorbed energy is transferred to the second component, the so-called quencher, according to the principle of fluorescence resonance energy transfer (FRET). During the annealing step of the PCR reaction, the hybridization probe binds to the target DNA and is degraded by the 5′-3′ exonuclease activity of the Taq polymerase during the subsequent elongation phase. As a result the excited fluorescent component and the quencher are spatially separated from one another and thus a fluorescence emission of the first component can be measured (U.S. Pat. No. 5,538,848).
Molecular Beacons: These hybridization probes are also labeled with a first component and with a quencher, the labels preferably being located at both ends of the probe. As a result of the secondary structure of the probe, both components are in spatial vicinity in solution. After hybridization to the target nucleic acids both components are separated from one another such that after excitation with light of a suitable wavelength the fluorescence emission of the first component can be measured (U.S. Pat. No. 5,118,801).
FRET hybridization probes: The FRET hybridization probe test format is especially useful for all kinds of homogenous hybridization assays (Matthews, J. A., and Kricka, L. J., Anal. Biochem. 169 (1988) 1-25). It is characterized by a pair of two single-stranded hybridization probes which are used simultaneously and are complementary to adjacent sites of the same strand of the amplified target nucleic acid. Both probes are labeled with different fluorescent components. When excited with light of a suitable wavelength, a first component transfers the absorbed energy to the second component according to the principle of fluorescence resonance energy transfer such that a fluorescence emission of the second component can be measured when both hybridization probes bind to adjacent positions of the target molecule to be detected.
When annealed to the target sequence, the hybridization probes must sit very close to each other, in a head to tail arrangement. Usually, the gap between the labeled 3′ end of the first probe and the labeled 5′ end or the second probe is as small as possible, i.e. 1-5 bases. This allows for a close vicinity of the FRET donor compound and the FRET acceptor compound.
Besides PCR and real time PCR, FRET hybridization probes and molecular beacons are used for melting curve analysis. In such an assay, the target nucleic acid is amplified first in a typical PCR reaction with suitable amplification primers. The hybridization probes may already be present during the amplification reaction or added subsequently. After completion of the PCR-reaction, the temperature of the sample is constitutively, i.e. continuously, increased, and fluorescence is detected as long as the hybridization probe was bound to the target DNA. At melting temperature, the hybridization probes are released from their target, and the fluorescent signal is decreasing immediately down to the background level. This decrease is monitored with an appropriate fluorescence versus temperature-time plot such that a first derivative value can be determined, at which the maximum of fluorescence decrease is observed. Alternatively it is possible to use a fluorescent-labeled primer and only one labeled oligonucleotide probe (Bernard, P. S., et al., Anal. Biochem. 235 (1998) 101-107).
Single label probe (SLP) format: This detection format consists of a single oligonucleotide labeled with a single fluorescent dye at either the 5′- or 3′-end (WO 02/14555). Two different designs can be used for oligonucleotide labeling, G-quenching probes and nitroindole-dequenching probes.
In the G-quenching embodiment, the fluorescent dye is attached to a C at the 5′- or 3′-end of the oligonucleotide. Fluorescence decreases significantly when the probe is hybridized to the target, in case two G's are located on the target strand opposite to C and in position 1 aside of the complementary oligonucleotide probe.
In the nitroindole dequenching embodiment, the fluorescent dye is attached to Nitroindole at the 5′- or 3′-end of the oligonucleotide. Nitroindole somehow decreases the fluorescent signaling of the free probe. Fluorescence increases when the probe is hybridized to the target DNA due to a dequenching effect.
In US patent application US 2003/0022177, Wittwer et al. principally introduced base analogs for the modification of the terminal ends of probe oligonucleotides. The corresponding labeled probe oligonucleotides showed the same performance as conventional oligonucleotide probes without base analogs, i.e. fluorescence increase upon hybridization with the target nucleotide sequence and fluorescence decrease after dissociation. Dequenching probes containing base analogs have been named but neither their synthesis nor their detailed characteristics have been described.
PCR products can be quantified in two fundamentally different ways.
End point determination of the amount of PCR product formed in the plateau phase of the amplification reaction: In this case the amount of PCR product formed does not correlate with the amount of the initial copy number since the amplification of nucleic acids at the end of the reaction is no longer exponential and instead saturation is reached. Consequently different initial copy numbers exhibit identical amounts of PCR product formed. Therefore the competitive PCR or competitive RT-PCR method is usually used in this procedure. In these methods the specific target sequence is coamplified together with a dilution series of an internal standard of a known copy number. The initial copy number of the target sequence is extrapolated from the mixture containing an identical PCR product quantity of standard and target sequence (Zimmermann, K., and Mannhalter, J. W., BioTechniques 21 (1996) 280-279). A disadvantage of this method is also that measurement occurs in the saturation region of the amplification reaction.
Kinetic real-time quantification in the exponential phase of PCR: In this case the formation of PCR products is monitored in each cycle of the PCR. The amplification is usually measured in thermocyclers which have additional devices for measuring fluorescence signals during the amplification reaction. A typical example of this is the Roche Diagnostics LightCycler (Cat. No. 2 0110468). The amplification products are for example detected by means of fluorescent labeled hybridization probes which only emit fluorescence signals when they are bound to the target nucleic acid or in certain cases also by means of fluorescent dyes that bind to double-stranded DNA. A defined signal threshold is determined for all reactions to be analyzed and the number of cycles required to reach this threshold value is determined for the target nucleic acid as well as for the reference nucleic acids such as the standard or housekeeping gene. The absolute or relative copy numbers of the target molecule can be determined on the basis of these values obtained for the target nucleic acid and the reference nucleic acid (Gibson, U. E., et al., Genome Res. 6 (1996) 995-1001; Bieche, I., et al., Cancer Res. 59 (1999) 2759-2765; WO 97/46707; WO 97/46712; WO 97/46714). Such methods are also referred to as a real-time PCR.
For synthesizing nucleic acid probes several compounds and their use for incorporation as monomeric units into nucleic acids are known in the art. Such compounds provide functional groups and/or linking moieties for the covalent attachment of reporter groups or labels. In the course of the chemical synthesis of the oligomeric compound, the skeletal structure of the “non-nucleotide compound” or “modified nucleotide” is connected with the “oligonucleotide” backbone, for example by phosphoramidite-based chemistry resulting in a phosphodiester. A given incorporated compound thus represents a modified nucleotide within the newly generated “modified oligonucleotide”. A label is bound by a functional group, exemplified by, but not limited to, an amino function that is present on the skeletal structure proper or on the “linking moiety”, which connects the skeleton with the functional group. A label can be covalently attached to the compound prior to the synthesis of a “modified oligonucleotide” or afterwards, upon the removal of an optional protecting group from the functional group to which the label is to be coupled.
EP 0135587 describes modifications of conventional nucleosides which carry a reporter group attached to a substituent group of the nucleotide base. EP 0313219 discloses non-nucleoside reagents characterized by a linear hydrocarbon skeletal structure with a linking moiety, or a side group, to which a label can be bound. EP 0313219 is silent about other types of skeletal structures and their particular properties.
U.S. Pat. No. 5,451,463 describes trifunctional non-nucleotide reagents, particularly 1,3-diol-based skeletal structures possessing a primary amino group. Such reagents can be used for example for terminal labeling of 3′ termini of oligonucleotides. WO 97/43451 discloses non-nucleotide reagents based on a carbocyclic (C5 to C7) skeletal structure, whereby a substituted or unsubstituted cyclohexane is preferred.
In summary, this technologies are either based on non-nucleosidic linkers/monomeric compounds which upon internally incorporation result in disruption of the probe structure or on modifications of a specific nucleobase, which requires for flexible internal labeling the synthesis of four different phosphoramidites.
Thus, it was the object of the present invention to overcome the afore described problems by providing an alternate labeling system which allows for an easy as well as position and dye independent labeling. In another aspect, the objective of the present invention was to provide improved probes for nucleic acid amplification, detection and quantitation.